In this Phase I program we will establish the feasibility of a novel filter-paper whole blood sample based targeted sequencing assay based on the TempO-Seq platform to measure DNA and RNA profiles as the basis for the development of a minimally invasive prognostic/diagnostic assay of Alzheimer?s Disease (AD). We will use our already functional whole transcriptome targeted gene expression assay to evaluate a set of whole blood samples from AD and normal donors provided by our collaborators, Dr. Allan Levey (Director, Alzheimer?s Disease Res. Center at Emory Univ., Chair, Dept. of Neurology) and Dr. Aaron Ritter (Cleveland Clinic, Lou Ruvo Center for Brain Health). We will utilize the resulting data to define a smaller targeted AD-specific gene subset, then redesign the probes for those genes to be compatible with the TempO-Seq DNA assay. This will allow us to generate a combined DNA/RNA assay capable of detecting both critical DNA genotypes (e.g. APOE alleles) and the diagnostic/prognostic gene expression RNA profile from dried blood spot samples, all in a single-well, addition-only assay, with no extraction, reverse transcription, or wash steps. Finally, we will combine the assay with a fill-in element, allowing mapping of significant stretches of genes which can harbor multiple unpredictable mutations (such as PSEN1/2 or APP), to create a ?one-shot? assay capable of detecting all relevant genetic and transcriptomic biomarkers in one single step, all from small (1-3 mm2) areas of blood spots on filter paper. The functionality of the completed assay will be confirmed on the same set of normal/AD samples, allowing us to proceed to Phase II, where we will commercialize the assay and prove its predictive and diagnostic value. This novel assay will address the unmet need for a cost-effective, simple way to broadly screen at-risk populations, especially in hard-to-reach or otherwise underserved areas. Ability to derive data of such unprecedented depth from samples that are as non-invasive and simple to collect and ship holds promise to reshape AD detection and care. The proposed research program is responsive to the NIA/DBSR Special Interest Topic D.1. to ?Develop multiple and reliable assays for limited blood-spot specimens for large surveys?, and Division of Aging Biology Topic J ?Development of biomarkers for prognosis, diagnosis, or treatment monitoring of aging or aging-related diseases?, and DN Topic A ??development of minimally-invasive biomarkers that can be used for screening in the general populations... biomarkers that could serve as surrogate measures for disease progression in MCI, AD and ADRD??. The outcome will be an assay that can be taken into Phase II to begin the development and validation of a prognostic/diagnostic screening assay for AD.
Blood spots collected on filter paper represent an easy-to-collect, easy-to-store, and easy-to-ship sample type, but one that also presents significant technical challenges for transcriptomics, as extraction from filter paper tends to produce low RNA yield and quality. Having already developed a gene expression assay capable of detecting RNA levels directly on small samples of blood on filter paper, we now propose development of a targeted sequencing assay which is performed directly on the filter paper sample itself, and which will produce a full readout of gene expression biomarkers correlated with Alzheimer?s Disease detection and progression. Furthermore, we propose combining this assay with a readout of relevant DNA genotypes (such ApoE4, or presenilin mutants), for a fully one-shot, low-cost assay amenable to high throughput processing.
